FIG. 2.

Regulation of endogenous LBH mRNA expression in human 293T cells by Wnt signaling. (A to C) qPCR analysis measuring relative mRNA levels of LBH, DKK1, and β-catenin normalized to GAPDH mRNA levels. All values represent means ± standard deviations (n = 3). (A) Time course analysis showing induction of LBH and DKK1 expression in response to Wnt3a. Cells were treated with Wnt3a-conditioned medium (Wnt3a) for the indicated time points. Wnt3a-mediated induction of both genes was inhibited by coadministration of recombinant Wnt inhibitor DKK1 (100 ng/ml), which was added 8 h prior to an 8-h treatment of cells with Wnt3a. (B) siRNA knockdown of β-catenin abolishes Wnt3a-induced upregulation of LBH, confirming activation of LBH expression by the canonical Wnt pathway. At 72 h posttransfection, 293T cells were treated with vehicle (−) or with Wnt3a-conditioned medium (+) for an additional 16 h. Note the reduction of β-catenin mRNA levels to less than 20% of endogenous expression levels in β-catenin (β-cat) siRNA-transfected cells. (C) Wnt7a, but not Wnt5a, inhibits Wnt3a-induced activation of LBH and DKK1. Cells were treated for 16 h with recombinant Wnt5a or Wnt7a proteins (100 ng/ml) individually or in conjunction with Wnt3a. As a control, cells were treated for 16 h with bovine serum albumin (BSA) vehicle.